Quantification and distribution of mast cells in oral periapical inflammatory lesions.

Background: Periapical granuloma and periapical cysts develop as a sequela of inflammation of dental pulp, usually a consequence of an infected root canal. Mast cells (MCs) have most frequently been implicated in the pathophysiology of periapical lesions such as periapical granulomas and cysts. Aims: The present study was undertaken to emphasize the presence and quantification of MCs in periapical lesions (granulomas and cysts) using toluidine blue stain, with an aim to elucidate their role in the pathogenesis of these lesions. Materials and
Methods: The study comprised a total of 55 previously diagnosed cases (30 cases of radicular cyst and 25 cases of periapical granuloma) selected from the departmental archives. The MCs were quantified in all the sections in the three zones, namely the epithelial, subepithelial, and deeper connective tissue zones. The results were sent for statistical analysis. Statistical Analysis: Wilcoxon paired t-test and Mann-Whitney U test were applied for intra- and intergroup comparison MCs in different zones of periapical granulomas and radicular cyst, respectively.
Results: A statistically significant difference was observed between the two lesions in all the three zones, i.e., epithelial, subepithelial, and in deeper connective tissue zone with P = 0.001.
Conclusion: Although MCs were present in all the three zones of both periapical cysts and granuloma, they were predominantly observed in radicular cysts than in periapical granuloma. This suggests a significant role of MC in initiation, development, and progression of these periapical lesions. Copyright:

INTRODUCTION

Mast cell (MC) is an eminent cell of the immune system, packed with numerous electron-dense metachromatic cytoplasmic granules, having a wide range of immunomodulatory functions in both healthy and diseased states. The primary roles of MCs are in allergy, asthma, and anaphylaxis, but they also play a significant role in protection, being intimately involved in wound healing and defense mechanism.[1]

MCs were initially illustrated by Paul Ehrlich in 1878 who named them as “mastzellen” meaning “feeding cells” due to their unique staining characteristics and presence of large granules.[2] MCs are present in proximity of blood vessels, and generally circumscribe the epithelium of the skin, mucosa of the lungs, and digestive tract along with oral cavity, conjunctiva, and nose.[34]

Initiation of periapical lesions is an outcome of chronic stimulation of periapical tissue as a result of percolation of inflammatory products from infected root canal. Bacterial antigens in its vicinity instigate host immune responses.[5] In view of recent evidence that MCs can synthesize a wide variety of mediators and are also responsible for reciprocal functional interaction between mast cell activation and subsequent T-cell stimulation. These findings suggest that mast cells can significantly modulate immune response in diverse physiological and pathological conditions. The present study is undertaken to compare and quantify MCs in different zones of periapical granuloma and cysts and thus to identify their role in the pathogenesis of the lesion.[6]

There is increased awareness regarding the contribution of MC in modulating immune response (humoral and cellular events).[7] They control vascular tone and vessel permeability by releasing secretary mediators such as histamine, serotonin, and other vasoactive amines. Mast cell have range of functions like they are the key players in regulating inflammatory response, involved in immunopathology of both immediate and delayed type of hypersenstivity reaction and they also play an axial role in amplification of host defense against bacterias and allergens. MCs synthesize a wide range of mediators, and hasten the cascade for early MC activation and sequential T-cell stimulation.[8]

The present study has been undertaken to provide a better understanding about the location and functional significance of MCs in disease progression in periapical granuloma and radicular cyst.

MATERIALS AND METHODS

The present retrospective study was carried out in the Department of Oral Pathology and Microbiology. The study was approved by the Institutional Ethical Committee (HCDSH/IEC/2018/046 dated February 19, 2018). A total of 55 previously diagnosed cases (30 cases of radicular cyst and 25 cases of periapical granuloma) with detailed clinical history were selected from the department archives. From all the paraffin blocks, two sections were cut each of about 4 to 5 μm in thickness. One set of section was stained with hematoxylin and eosin to confirm the clinical diagnosis and the other set of section was stained with 1% toluidine blue stain for identification of MCs.

The MCs were counted in the stained sections using binocular light microscope with ×10 eyepiece fitted with a 1 cm2 graticule and high-power (×40) magnification. The area encircling 1 cm2 by the graticule was kept as one microscopic field, and the MCs confining within the zone were summed. MC counting was performed in three zones in all sections. The graticule was oriented along the epithelium (epithelial zone) and the fields were selected randomly, and the counting was performed in a step ladder fashion proceeding along the entire length of the section. The graticule was then moved to the subepithelial zone, and the counting was repeated again in a similar manner. Later, the graticule was moved down into the deeper connective tissue, and the counting was repeated similarly [Figures 1 and 2].

Figure 1

Photomicrograph showing numerous mast cells in periapical granuloma (toluidine blue stain; low power)

Figure 2

Photomicrograph showing numerous mast cells in radicular cyst (toluidine blue stain; high power)

The MCs counted in different fields are later added to get the total count of MCs in the particular field. The results thus obtained by the above method were then tabulated and sent for statistical analysis using the “Mann–Whitney U test” for the intergroup comparison and “Wilcoxon signed-rank test” for intragroup comparisons.

RESULTS

When the number of MCs was quantified in 25 cases of periapical granuloma using Friedman test, Chi-square value came out to be 50.00 and P = 0.0001* (statistically significant). The result suggests that MCs were numerous in subepithelial zone than in deeper connective tissue and least in epithelial zone. On intergroup comparison between radicular cyst and periapical granuloma using Mann–Whitney U-test at P < 0.05, it was observed that MC count was significantly higher in radicular cyst compared to periapical granuloma for epithelial, subepithelial, and deeper connective tissue zone with P = 0.001* (statistically significant) [Table 1 and Graph 1].

Table 1

Intergroup comparison of the mast cell counts in different zones of periapical granuloma and radicular cyst

Grouping variable	n	Mean	SD	Z	P
Epithelial
PG	25	7.24	1.01	−3.463	0.001*, significant
RC	30	8.50	1.17
Total	55	7.92	1.25
Subepithelial
PG	25	15.92	1.75	−5.182	0.001*, significant
RC	30	19.67	2.15
Total	55	17.96	2.72
Deeper connective tissue
PG	25	12.52	1.73	−6.367	0.001*, significant
RC	30	24.77	2.19
Total	55	19.20	6.46

Mann-Whitney U test, level of significance at P<0.05. SD: Standard deviation, PG: Periapical granuloma, RC: Radicular cyst, *As discussed with the statistician the significance value is same for all the groups

Graph 1

Mast cell count in different zones of periapical granuloma and radicular cyst

On pairwise comparison of all the three zones of periapical granuloma (using Wilcoxon paired t-test), significant differences in the count of MCs were found in all the three at P < 0.05. Similarly, when the number of MCs was quantified in 30 cases of radicular cyst, Chi-square value came out to be 60.00 and P = 0.0001* (statistically significant). On zone-wise distribution, MC count was higher in deeper connective tissue than in subepithelial zone and least in epithelial zone. On pairwise comparison in all the three zones of radicular cyst, significant differences in the count of MCs were found in all the three at P < 0.05 [Table 2].

Table 2

Pairwise comparison of mast cell counts in different zones of periapical granuloma and radicular cyst

Zones	Subepithelial - Epithelial		Deeper connective tissue - Epithelial		Deeper connective tissue - Subepithelial
	PG	RC	PG	RC	PG	RC
Z	−4.406b	−4.802b	−4.429b	−4.813b	−4.430c	−4.853b
P	0.0001*		0.0001*		0.0001*
	Highly significant		Highly significant		Highly significant

Wilcoxon paired t-test, level of significance at P<0.05. PG: Periapical granuloma, RC: Radicular cyst. *As discussed with the statistician, the significant values are same as 0.0001 for all the groups. Superscript b is a constant, which signifies paired t test which has been applied in pair wise comparision between different epithelial zone3s. Superscript c at one place is a misprint error, there also the superscript should be b

DISCUSSION

In response to bacterial antigens, a cascade of host immune responses is stirred, including antigen-specific immunologic responses and nonspecific inflammatory reactions.[9] MCs have been found in large numbers in the periapical inflammatory tissues of granulomas and cysts, advocating a potential role played by them in modulating cellular immune mechanisms of these lesions.[5]

In sequel to pulp necrosis, the periapical granuloma begins when bacterial antigens in the root canal stimulate an immunological reaction in the periapical tissues. The low-grade irritation leads to a chronic process followed by hyperemia and edema of the periodontal ligament with infiltration of chronic inflammatory cells.[1011] As a consequence of these inflammatory processes, there is increased vascularity which results in resorption of periapical bone and as well at times resorption of the tooth apex.[12] Radicular cysts are thought to arise as a consequence of inflammation, resulting in stimulation of epithelial rests residing within periodontal ligament.[13]

In the present study, on quantitative analysis of MC in periapical granuloma, the mean MC count was reported as 7.24, 15.92, and 12.52 in the epithelial, subepithelial, and deeper connective tissue zone, respectively, indicating that MCs are more prevalent in the subepithelial zone than in the deeper connective tissue and less prominent in the epithelial zone. The mean MC count in the above three zones in radicular cyst was reported as 8.5, 19.67, and 24.77, respectively, in contradiction to periapical granuloma, radicular cyst showed higher mean MC density in deeper connective tissue zone than in subepithelial zone and least in epithelial zone. On pairwise comparison between all the three zones (epithelial zone, subepithelial zone, and deeper connective tissue zone), the difference was statistically significant (P = 0.0001*) for both the lesions.

Many studies in literature have claimed that MCs play an active role in the initiation and development of periapical inflammatory lesions.[14] Several authors have dissimilar opinions regarding the location of MC in different areas of the lesion, some suggest that MC is predominant in inflammatory areas, few say that they are chiefly located in a perivascular position, while Kontianen et al. reported predominant MC in fibrotic areas.[3141516] Various studies have reported MCs in different zones of tissue in inflammatory periapical lesions, signifying their role in the initiation, development, and progression of such lesions. MCs have been linked to various steps in inflammatory process such as vasodilation, angiogenic response, proteoglycan production, collagen synthesis, initiation of inflammation, bone resorption, and destruction of extracellular matrix.[211121317]

MCs are closely associated with immunologic, physiologic, and pathologic processes of inflammation.[18] It has been already established that an interaction between MC and T-lymphocytes causes liberation of histamine from MCs, inhibiting T-lymphocyte activity against antigens.[1920] Dohlsten et al. noticed that histamine prevents the production of IL-2 and gamma-interferon.[21] Piatelli et al. observed that MCs are positive to IL-2 receptor and recommended that these cells create a negative feedback mechanism in controlling the immunologic reaction, by preventing IL-2 stimulation and thereby fixing it in their membranes.[22] Our results support this theory by sustained presence of numerous MC in areas of chronic inflammatory infiltrate, suggesting that MC could play an active role in commencement, development, and persistence of the inflammatory process in allied periapical lesions.

MCs contain few specific proteolytic enzymes, namely tryptase and chymase, which play an essential role in the breakdown of proteoglycan of the connective tissue capsule of jaw cysts in normal and in inflammatory conditions, thus causing degradation of extracellular matrix.[1323] Few studies have reported the active involvement of MC tryptase in bone tissue remodeling in odontogenic jaw cysts.[24] The MCs contribute to cystic enlargement by increasing the intraluminal hydrostatic pressure by three processes; firstly, by releasing heparin into the luminal fluid; secondly, causing degradation of extracellular matrix by secreting proteolytic enzymes, thus facilitating their passage into the fluid; and finally, by the action of histamine on smooth muscle contraction and vascular permeability which enhances transudation of serum proteins. MCs have also been implicated in initiating the production of prostaglandins which may cause bone resorption and thereby assisting in cyst enlargement.[311]

On the basis of immunohistochemical studies, Teronen et al. and Netto et al. have reported a higher frequency of MCs at the periphery of the cysts in proximity to perilesional bone and also adjacent to inflammatory cells. They speculated that MC participation in the inflammatory process is related to bone destruction, thereby causing enlargement of inflammatory periapical lesions.[511] The finding of the above studies is in accordance with the present study.

The present study contradicts the findings of the study conducted by Smith et al. in which they have stated that MCs are more prevalent just beneath the epithelium than in deeper areas of the cyst.[13] A histochemical study has reported a subepithelial banding or pattern of Alcian blue staining due to the presence of heparin, probably as a result of MC degranulation at these sites.[5] Degranulation of MCs and movement of intact MCs through the epithelium account for the presence of glycosaminoglycan in cystic fluids. This creates a chemotactic stimulus, thus attracting MCs to epithelial lining or luminal fluid contents of odontogenic cyst.[121319] The above findings conflict with the data reported by Teronen et al., Kontianen et al., and Netto et al. who reported that MCs are more frequent in fibrotic areas.[111624]

On comparing the mean MC count in periapical granuloma and radicular cyst, it was found that radicular cyst showed a higher count than periapical granuloma. The differences in the three zones were statistically significant (P < 0.001). This is in accordance with studies conducted by authors such as Perrini and Fonzi and Moon et al. who have also emphasized the significance of MCs in the pathogenesis of chronic inflammatory lesions.[1520]

CONCLUSION

Periapical granuloma and radicular cyst are recognized as one of the most common inflammatory periradicular reactions in response to microbial and chemical stimuli approaching from the pulp. On comparing both the lesions, a higher total MC count was observed in radicular cyst than periapical granuloma. This explains that MC plays an important role in the commencement and development of chronic periapical inflammatory lesions. This study opens a gateway for further researches to be undertaken to evaluate the influence of MC antagonists on cyst expansion and to unravel the precise role played by them in the pathogenesis of other inflammatory lesions and odontogenic cysts too.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.